1. Field of the Invention
The invention relates generally to the field of induced pluripotent stem cells (iPSCs) and more specifically to methods of generating such cells, as well as identifying agents useful in generating iPSCs.
2. Background Information
Embryonic stem (ES) cells are not only versatile tools for investigating early developmental events but provide a promising source of tissues potentially useful for regenerative therapies. Recent breakthroughs in generating iPSCs provide alternative means to obtain ES-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. iPS cells are known to share numerous traits with ES cells, such as colony morphology, transcriptome, self-renewal ability and pluripotency. Moreover, customized therapeutic applications of iPS cells have been reported. Nonetheless, the molecular basis of reprogramming remains unclear.
Reprogramming is a step-wise process moving from differentiated to ES-like stages, a progression that can be monitored using various cellular markers. The differentiation marker, Thy1, is highly expressed in mouse embryonic fibroblasts (MEFs), and its expression in MEFs decreases within a few days of transduction with Oct3/4, Sox2, Klf4, and c-Myc (denoted here 4F: OSKM). Consequently, expression of the stem cell marker SSEA1 increases, followed by activation of other ES markers, such as endogenous Nanog, Oct3/4, and X reactivation. During this process, iPS cells have been shown to be enriched or selected. Increasing evidence indicates that the four reprogramming factors cooperatively initiate the transition of cell identity from somatic to iPS cells.
These data suggest that signature patterns of gene expression in MEFs constitute a barrier for induced reprogramming and that overcoming this barrier may be a rate-limiting step in the reprogramming process. Despite advances in reprogramming, efficiency of reprogramming remains low and inefficient, thus new approaches are necessary to generate iPSCs.